Abstract PD10-03: PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC)
Authors: Chia Chia Liu, Lanfang Qin, Shanunak Sathe, Sarmistha Nanda, Jamunarani Veeraraghavan, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C. Kent Osborne, Xiaoyong Fu, Rachel Schiff
Journal: Cancer Research (2023)

Abstract Background: Recent studies suggest that enhancer reprogramming underlies heterogeneity and disease progression in estrogen receptor-positive (ER+) BC. Cell-type/state specific transcription is governed by high-order assemblies of master transcription factors (TFs) and epigenetically defined regulatory regions including super-enhancers (SEs). We previously showed that aberrant activation of the pioneer TF FOXA1 promotes enhancer and transcriptional reprogramming in endocrine-resistant BC, involving the ER and the AP-1 FRA1 and c-JUN TFs. As SEs maintain a robust cell-type/state specific core transcriptional regulatory circuitry (CRC) in developmental and tumorigenic processes, we sought to identify key additional TFs in SE/FOXA1-driven CRCs in endocrine resistance, which could serve as attractive therapeutic targets. Methods: TF binding motif at the shared SEs (mapped by H3K27ac ChIP-seq) between MCF7-parental (P) cells with ectopic FOXA1 overexpression (OE) and the endogenous FOXA1-amplifed tamoxifen-resistant (TamR) cells was analyzed by HOMER. ER-bound SEs distinguishing TamR vs. P cells were defined by integrating the SEs with our prior ER ChIP-seq data (PMID 28507152). KLF4 motif within these ER-bound SEs was scanned using FIMO and linked to nearby genes by intersection with the previously defined promoter-tethered regions (PTRs) (PMID 24141950). Differential gene expression in MCF7-TamR cells upon KLF4 knockdown (KD) by 3 unique siRNAs was analyzed using limma from edgeR. The biological and clinical significance of the KLF4-dependent genes was analyzed using Gene Ontology and survival modeling with METABRIC and the ER+ metastatic BC cohort (SABCS19-GS2-02). Cell migration was assessed by the wound-healing assay. Results: We identified KLF4 among the top enriched TF binding motifs at the shared SEs in FOXA1-overexpressing MCF7-P cells and the FOXA1-amplified TamR cells. Analysis of our prior RNA-seq data of MCF7-P and TamR cells upon OE or siRNA KD of FOXA1, FRA1, or c-JUN (PMIDs 27791031, 31826955, 32424275, SABCS21-PD1-05) revealed KLF4, the Yamanaka factor for induced pluripotent stem cells, as a common target activated by the FOXA1/FRA1/c-JUN axis. We next identified 44 genes commonly down-regulated upon KLF4 KD in the MCF7-TamR cells. This KLF4-dependent 44-gene set was enriched in biological processes of embryonic development and tumor progression, preferentially dependent on ER in MCF7-TamR vs. P cells, highly elevated in ER+ metastases vs. primary tumors, and associated with poor outcome in ER+ BC treated with endocrine therapy. KLF4 KD, using the 2 siRNAs that generated similar pathway perturbations in MCF7-TamR cells, reduced TamR cell migration. Notably, among the genes co-dependent on KLF4 and ER in TamR cells, PYGB was the only gene with a PTR residing in an ER-bound SE established in TamR but not P cells. Glycogen phosphorylase B, encoded by PYGB, is the rate-limiting enzyme in glycogen degradation and plays a role in the progression of various tumors. Expression of KLF4, FOXA1, and FRA1 are commonly activated during the differentiation of human embryonic stem cells into foregut endoderm and in the inner core of fibroblasts of first-trimester human placenta villi, suggesting a unique role of KLF4 in mediating lineage-specific CRC, possibly by engaging PYGB and the glycogen metabolic pathway in advanced ER+ disease. Conclusions: Using SE-oriented integrative bioinformatics, we identified KLF4 as a potential novel target in the FOXA1/AP-1 transcriptional axis. As KLF4 binding motif resides in the unique ER-bound SEs of TamR cells, KLF4 likely forms an auto-regulated loop amplifying CRC in transcriptional reprogramming, among which the PYGB/glycogen metabolic pathway merits further investigation in endocrine-resistant ER+ disease. Citation Format: Chia Chia Liu, Lanfang Qin, Shanunak Sathe, Sarmistha Nanda, Jamunarani Veeraraghavan, Ofir Cohen, Nikhil Wagle, Mothaffar Rimawi, C. Kent Osborne, Xiaoyong Fu, Rachel Schiff. PD10-03 Super-enhancer-oriented integrative bioinformatics identifies aberrant KLF4 signaling in endocrine-resistant breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-03.